Biochemical and Biological Properties of Myosins

我们感兴趣的是肌球蛋白I类和II类肌球蛋白肌动蛋白激活的ATPase活性调节的分子基础，这些肌球蛋白的生物学作用及其生物活性的调节。在一个项目中，为了了解肌球蛋白头部和尾部对酶活性和功能的耦合作用，我们研究了由棘阿米巴或平滑肌肌球蛋白II的运动区和尾区组成的嵌合体的性质。嵌合体的肌动蛋白激活的ATPase活性比野生型Dictyostelialmyosin II高10-15，但与野生型肌球蛋白相反，这种活性既不受调节轻链的磷酸化调节，也不受棘阿米巴嵌合体尾部的磷酸化调节。当在肌球蛋白II缺失的细胞中表达时，这两个嵌合体都拯救了两种肌球蛋白II依赖的功能，悬浮培养中的胞质分裂和conA受体的封顶，但不支持第三种肌球蛋白II依赖的功能，即充分发育到子实体。此前已有研究表明，Dictyostelialmyosin II的尾部结构域包含定位到分裂细胞的分裂沟(Cf)所需的所有信息。我们现在已经发现，在体外组装的最短的尾部片段-283个残基组装结构域在肌球蛋白II缺失的细胞中表达时会进入Cf，棘阿米巴肌球蛋白II尾部的256个残基区域也是如此。因此，似乎没有特定的序列要求将肌球蛋白定位到Cf。我们比较了Dictyostelialmyosin II的HCM/TEDS位点表面环的15个突变体支持肌球蛋白II依赖的三种功能的能力。鸡的平滑肌肌球蛋白环支持正常的conA受体封顶，80%的正常生长和轻微的发育受损。人类β-心肌肌球蛋白环仅支持野生型的25%的生长，不完全封顶和严重损害发育，即到倾斜丘状阶段，仅略好于肌球蛋白II缺失的细胞。各种点突变使平滑肌环功能完全发挥作用，而心脏环的功能更像是平滑肌环。所有突变体的肌动蛋白激活的ATPase和体外运动活性正在研究中，以确定是否与它们的生物学特性相关。棘阿米巴和肌球蛋白I的重链头部、颈部和尾部的重链结构域和轻链以不同的组合互换的嵌合体的研究为肌球蛋白活性的三个重链结构域和轻链的相互依赖提供了进一步的证据。

We are interested in the molecular bases of the regulation of the actin-activated ATPase activities of class-I and class-II myosins, the biological roles of these myosins and the regulation of their biological activities. In one project, to understand the coupled contributions of myosin heads and tails to enzymatic activity and function, we studied the properties of chimeras consisting of the motor domain of Dictyostelium myosin II and the tail domains of either Acanthamoeba or smooth muscle myosin II. The chimeras have 10-15 higher actin-activated ATPase activity than wild-type Dictyostelium myosin II but, in contrast to wild-type myosins, this activity is not regulated by phosphorylation of either the regulatory light chain or, in the Acanthamoeba chimera, phosphorylation of the tail. When expressed in myosin II-null cells, both chimeras rescue two myosin II-dependent functions, cytokinesis in suspension culture and capping of con A receptors, but do not support a third myosin II-dependent function, full development to fruiting bodies. It had previously been shown by others that the tail domain of Dictyostelium myosin II contains all the information needed for localization to the cleavage furrow (CF) of dividing cells. We have now found that the 283-residue assembly domain, the shortest tail segment that assembles in vitro, goes to the CF when expressed in myosin II-null cells, as does also an equivalent 256-residue region from the tail of Acanthamoeba myosin II. Thus, there seems to be no specific sequence requirement for localization of myosin to the CF. We have compared the abilities of 15 mutants of the HCM/TEDS-site surface loop of Dictyostelium myosin II to support the three myosin-II dependent functions. The chicken smooth muscle myosin loop supports normal con A-receptor capping, 80% of normal growth and only slightly impaired development. The human beta-cardiac myosin loop supports growth at only 25% of wild-type rate, incomplete capping and greatly impaired development, i.e. to the the tipped mound stage, only slightly better than myosin II-null cells. Various point mutations make the smooth muscle loop fully functional and the cardiac loop function more like the smooth muscle loop. The actin-activated ATPase and in vitro motility activities of all of the mutants are being studied to see which, if either, correlates with their biological properties. Studies of chimeras in which the heavy chain head, neck and tail domains and light chains of Acanthamoeba and Aspergillus myosin I are interchanged in various combinations provide further evidence for the interdependence of the three heavy chain domains and light chains for myosin activities.